diagnosis of bartonella spp. infection: study of a ... · erythema nodosum or erythema multiformis,...

Received on January 28, 2003.Approved by the Consultive Council and accepted for publication on October 17, 2003.* Work done at Faculdade de Ciências Médicas da Universidade Estadual de Campinas / FCM - UNICAMP - Campinas (SP), Brazil.Conflict of interests: None

1 M.D., PhD, Professor of Dermatology at the Department of Internal Medicine, Faculdade de Ciências Médicas da Universidade Estadual de Campinas / FCM - UNICAMP - Campinas (SP), Brazil.


3 M.D., PhD, Professor, Head of the Departament of Pathological Anatomy at the Faculdade de Ciências Médicas da Universidade Estadual de Campinas / FCM - UNICAMP - Campinas (SP), Brazil.



©2006 by Anais Brasileiros de Dermatologia

Diagnosis of Bartonella spp. infection: study of a bacillaryangiomatosis case*

Diagnóstico da infecção por Bartonella spp.: a propósito deum caso de angiomatose bacilar*

Paulo Eduardo Neves Ferreira Velho1 Elemir Macedo de Souza2 Maria Letícia Cintra3

Aparecida Machado de Moraes4 Ana Maria Uthida-Tanaka5

Abstract: Several dermatoses are considered idiopathic diseases. Many times lesions such aserythema nodosum or erythema multiformis, for example, cannot have their etiology defined.Human infection caused by Bartonella spp. may determine several clinical syndromic expres-sions. Starting with a clinically, histologically and ultrastructurally documented bacillaryangiomatosis case, a review of medical literature was undertaken to evaluate the availablediagnostic criteria regarding infection caused by these agents. Histological evaluations wereconcluded to be, practically speaking, an important and useful diagnostic method, especiallywhen serology is not available.Keywords: Bartonella; Diagnosis; Skin

Resumo: Várias dermatoses são consideradas idiopáticas. Muitas vezes, afecções comoeritema nodoso ou eritema multiforme, por exemplo, não podem ter sua etiologia definida.A infecção humana por Bartonella spp. pode determinar várias expressões clínicas sin-drômicas. A partir de um caso de angiomatose bacilar com documentação clínica, his-tológica e ultra-estrutural, foi feita a revisão da literatura médica para avaliar os critériosdiagnósticos disponíveis para a infecção por esses agentes. Conclui-se que a avaliação his-tológica é, na prática, um importante e útil método diagnóstico, especialmente quando asorologia não estiver disponível.Palavras-chave: Bartonella; Diagnóstico; Pele

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Case Report349

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FIGURE 1: Bacillary angiomatosis and Norwegian scabies. A: Scalpdiffuse pilous rarefaction and angiomatous lesion; scale crusts onthe back. B: Angiomatous papular-tumoral lesion and scaling sur-

ronding skin

INTRODUCTIONBartonella spp. form a fascinating group of

pathogens and are responsible for emerging bacterialinfectious diseases. They cause Carrion’s disease, withits febrile (Oroya’s fever) and tecidual (verruga peruana)stages; trench fever, cat-scratch disease and bacillaryangiomatosis. Relapsing febrile bacteremias, endocardi-tis, septicemias, neurological, psychiatric, ophtalmologi-cal, osteus and hematologic manifestations are also asso-ciated with infection by these agents. Severe hemolyticanemia is a milestone of the febrile and immunosup-pressing stage of Carrion’s disease. Moreover, manyhuman bartonella infections have blood-sucking ectopa-rasites as vectors: lutzomites, lice and ticks.1

CASE REPORTForty-nine year-old brown female patient, who

had been diagnosed with AIDS two years before, hadbeen presenting for one month bleeding and painfulpapular and nodular-papular lesions, initially inupper limbs, later disseminating through the entirebody surface, including genitals, together with fever,weight loss, lack of appetite and malaise. Six monthsbefore, she had presented pruritic desquamativelesions in the entire body and mentioned having hadcontact with various cats.

Upon physical examination, signs of malnouris-hment, desquamation and squamous-crusts in trunk,especially back (Figure 1A), limbs and scalp wereobserved. Angiomatous papular and nodular-papularlesions (Figure 1B) were observed in the whole body,ranging from few millimeters to approximately 1.5cmin diameter, besides mild hepatosplenomegaly. Directscale examination was positive for acarides, sugges-ting Sarcoptes scabiei. Biopsies were taken to ruleout bacillary angiomatosis and tetracycline was pres-cribed orally in the dose of 2g/day.

On histological examination in hematoxylin-eosin staining, thinned epidermis and numerous der-mal capillaries with edematous-sometime atypical,endothelial cells were observed (Figures 2A and 2B).There were frequent endotelial mitoses and neutro-philic inflammatory infiltrate. Whartin-Starry stainingshowed several cocobacilli inside macrophages andfree bacterial aggregates in interstitium (Figures 3Aand 3B), better demonstrate in epoxy-embedded tis-sue sections staining with toluidine blue (Figura 4A).Tripe laminar walled bacilli were easily identified ontransmission electron microscopy (Figure 4B).Improvement of fever and angiomatous lesionsoccurred after few days of treatment (Figures 5A and5B). On the twentieth day of hospitalization, patientwas discharged with clinical improvement of bothbacillary angiomatosis and scabies. Ambulatorial fol-low-up was lost.

DISCUSSIONBacillary angiomatosis is more frequent in

immunocompromised patients, especially those withAIDS, but can also affect immunocompetent indivi-duals. It is caused by Bartonella henselae and by B.quintana.2 The cat can serve as reservoir, but its roleseems not to be obligatory.3 Lesions are characterizedby capillary proliferation arranged in lobules.Cutaneous manifestations are the most frequent.Lesions can be papular, tumoral or nodular, multiple,single, and may be spread throughtout the entire

350 Velho PENF, Souza EM, Cintra ML, Moraes AM, Uthida-Tanaka AM.

An Bras Dermatol. 2006;81(4):349-53.

FIGURE 2: Bacillary angiomatosis. A: Hyperkeratotic and hiperplas-tic epidermis corresponding to the collarette of the lesion, charac-terized by proliferation of lobularly-disposed capillaries (HE, orig-

inal magnification x 100) B: Between swelled endothelial cells,scarce amorphic acidophilic (fibrinoid/arrow) material is deposited,were bacterial aggregates can be demonstrated by silver impreg-

nation (HE, original magnification original x 400)


An Bras Dermatol. 2006;81(4):349-53.

body.4 The main differential diagnosis is Kaposi’s sar-coma, with which it may coexist. Bacillary peliosis ischaracterized by angioproliferating lesions associatedto capillary dilation, formation of cavernous spacesfilled with blood in the liver, spleen or bone mar-row.5,6

Prompt evolution without treatment is poten-tially lethal, but usually presents rapid response toantibiotic therapy with erythromycin or doxicyclin.Treatment can be prolonged, going from two to fourmonths, in order to decrease the risk of relapse.7

Diagnosis is based on:Direct blood examination: blood smear, stai-

ned by Giemsa method, is useful for diagnosingOroya’s fever and for follow-up of patients infectedwith B. bacilliformis.8

1. Febrile conditions, especially those associa-ted with anemia, can have their etiologies definedwith this diagnostic test.

2. Microbiological examination: isolation of dif-ferent bartonella species usually requires prolongedtime, from two to six weeks of culture, using enrichedand recently prepared media, in a CO2-rich environ-ment. They have been isolated from blood samplesand tissue fragments, using both fresh and frozenmaterial.1 Brenner et al., 1997, showed that freezingblood sample collected with anti-coagulants at -65ºCfor 24h increased the number of seeded colony-for-ming units/mL, increasing sensitivity of the exam com-pared to direct plating in lysis tubes.9 Hemoculture ismore sensitive than techniques of genetic detection.However, neither blood or tissue bartonella culture isroutinily undertaken in most clinical pathology labora-

tories, both for the prolonged culture time and for thespecial conditions that are necessary for bacterialgrowth.

3. Serologic testing: immunoenzymatic, director indirect immunofluorescence assays for B. hense-lae and B. quintana have been the most widely usedtests for the diagnosis of Bartonella spp. infection.Nevertheless, the utilization of these tests for acutephase infections has been a focus of debate in litera-ture. For Baneth et al., 1996, it is likely that there is agreat antigenic diversity among species, subspeciesand strains of bartonellae.10 This can justify large dif-ferences found between serologic surveys from diffe-rent regions, especially when commercially availabletest kits are used.11 There seems to be no cross reac-tion among strains of B. henselae, neither among spe-cies of bartonella.12 Velho et al., 2002, after evaluatingthe ultra-structure of a standard strain of B. henselaeafter passings in mice, postuleted that, besidesgenotypic differences, also phenotypic and possiblyantigenic could justify the inconsistent serological fin-dings in the diagnosis of infections by these bacteria.13

4. Molecular biology genetic detection techni-ques: these methods have the goal of aiding in thediagnosis of Bartonella spp. infection, and also inepidemiological studies of strains and species of thisgenus or in genotyping. Its identification is based on

Diagnosis of Bartonella spp. infection: study of ... 351

FIGURE 3: A: Biopsy sample of a bacillary angiomatosis combinedwith Norwegian scabies lesion (silver impregnation): arrow points

at part of an egg of Sarcoptes scabiei (Whartin-Starry, originalmagnification x 100) B: Bacterial agglomerate interstitium

(Whartin-Starry, original magnification x 400)

FIGURE 4: A: Epoxy-embedded tissue semi-thin section (approxi-mately 300 nm) toluidine blue stain: several cocci-bacillary struc-

tures are seen in the interstittium, next to a capillary (originalmagnfication x 200) B: Bartonella can be easily individualized

(transmission eletron microssopy, original magnification x 16.000).Inset. Bacillus with triple laminar wall and numerous fimbriae

(original magnification x 80.000)

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An Bras Dermatol. 2006;81(4):349-53.

352 Velho PENF, Souza EM, Cintra ML, Moraes AM, Uthida-Tanaka AM.

molecular characterization in tissue culture obtainedfrom skin biopsy of .lymph node aspiration. Bacteriathat grow from hemocultures of patients with endo-carditis or fever can be used, as well as tissue samplesor bacterial cultures stored in laboratories.1

Intergenic region 16S-23S rRNA, used by Jensen et al.,2000, was shown to be an appropriate target for con-taining regions with genetic sequences that are diver-gent enough for allowing species differentiation. Themore colony-forming units/mL, the greater the sensi-tivity for genetic detection, reaching 100% of positi-vity when bacteremia levels were of 50-100units/mL.14

5. Microscopy: it is of great usefulness for casesof bacillary angiomatosis, verruga peruana and cat-scratch disease. The former two are histologicallyindistinguishable. For experienced pathologists, diag-nosis can be defined with basis on hematoxylin-eosinstaining for most cases. It is characterized by endo-thelial proliferation with lobular arrangement, withmore differentiated central vessels and less matureperipheral vessels, sometimes with their lumens

unapparent. Lesions remind piogenic granuloma,both clinically and histologically. Acute and chronicinflammatory infiltrate, even in non-ulcerated lesions,with leukocytes and neutrophils inside the lobules,are findings that suggest the diagnosis. Inflammatoryinfiltrates are found surrounding bacterial aggregates,which have a fibrin-like aspect when staining. Cellularatypies can be found, especially in more solid lesions,with unapparent lumens and fusiform cells remin-ding Kaposi sarcoma.1 In cat-scratch disease, histolo-gical findings are similar in primary lesion and affec-ted node, usually being constituted by granulomaswith necrotic cores, surrounded by lymphocytes andhistiocytes and with neutrophilic infiltrate, formingmicroabscesses. In the nodes, findings can be mista-ken with those of Hodgkin’s disease.15 Bacilli are seenwithin the initial neutrophilic inflammatory infiltrateand in recently formed granulomas, being rare withinmature ones. The same findings are obtained inepoxy-embedded tissue, semi-thin sections stainingwith toluidine blue. Transmission electronic micros-copy allows individualization of triple walled agents.Immunofluorescence and immunohistochemicaltechniques allow differentiation among bartonellaspecies.1

There is no “golden standard” for the diagnosisof bartonella infections. Serology is commercializedonly for research, and genetic detection techniquesare not available for clinical use in most medical cen-ters in the world.

Sensitivity for a given method is a function ofboth clinical manifestation and sample origin.4,9

Histopathological examination is important forcases of bacillary angiomatosis, verruga peruana andcat-scratch disease.

In granulomatous cases of undefined etiology,this differential diagnosis should be considered, espe-cially when microabscesses are present.

Actually, little is known about infection bythese bacteria. However, it is possible that many idio-pathic dermatoses are, in fact, caused by these agents.Man, cat, rat, mouse and rabbit can all live asympto-matically with bacteria from this genus. Their trueimportance for the development of diseases is unk-nown; it is certain, nevertheless, that bartonella infec-tions are potentially lethal, which justifies largerefforts in their study. �

FIGURE 5: Bacillary angiomatosis and Norwegian scabies. A:Lesions in the genital region and proximal thigh. Observe lesion

in right thigh before antibiotic treatment. B: Patient in theeleventh day of treatment with 2g/d tetracycline


REFERENCES1. Velho PENF. Estudo das bartoneloses humanas e da

Bartonella henselae: infecção experimental, microbiologiae microscopia de luz e eletrônica de transmissão[dissertação]. Campinas (SP): Faculdade de Ciências Médicas da Universidade Estadual de Campinas; 2001.

2. Tappero JW, Mohle–Boetani J, Koehler JE, Swaminathan B, Berger TG, Leboit PE, et al. The epidemiology of bacillary angiomatosis and bacillary peliosis. J Am Med Assoc. 1993;269:770-5.

3. Koelhler JE, Glaser CA, Tappero JW. Rochalimaea henselaeinfecion: a new zoonosis with the domestic cat as reservoir.J Am Med Assoc. 1994;71:531-5.

4. Levell NJ, Bewley AP, Chopra S, Churchill D, French P, Miller R, et al. Bacillary angiomatosis with cutaneous and oral lesions in an HIV-infected patient from the UK. Brit J Dermatol. 1995;132:113-5.

5. Loutit JS. Bartonella infections. Cur Clin Top Infec Dis. 1997;17:269-90.

6. Raoult D. Infections humaines à Bartonella. Presse Med. 1999;28:429-34.

7. Maurin M, Raoult D. Bartonella (Rochalimaea)quintana infections. Clin Microbiol Rev. 1996;9:273-92.

8. Garcia-Caceres U, Garcia FU. Bartonellosis: animmunodepressive disease and the life of Daniel Alcides Carrión. Am J Clin Pathol. 1991;95(Suppl 1):S58-66.

9. Brenner SA, Rooney JA, Manzewitsch P, Regnery RL. Isolation of Bartonella (Rochalimae) henselae: effects of methods of blood collection and handling. J Clin Microbiol. 1997;35:544-7.

10. Baneth, G, Kordick DL, Hergarty BC, Breitschwerdt EB. Comparative seroreactivity to Bartonella henselae and Bartonella quintana among cats from Israel and North Carolina. Vet Microbiol. 1996;50:95-103.

11. Dehio C, Sander A. Bartonella as emerging pathogens.

Trends Microbiol. 1999;7:226-8.12. Regnery RL, Rooney JA, Johnson AM, Nesby SL,

Manzewitsch P, Beaver K, et al. Experimentally induced Bartonella henselae infections followed by challenge exposure and antimicrobial therapy in cats. Am J Vet Res. 1996;57:1714-9.

13. Velho PENF, Moraes AM, Uthida-Tanaka AM, Cintra ML, Gilioli R. Ultrastructural changes in a standard strain of Bartonella henselae after passages through BALB/cAn mice. Ultrastruct Pathol. 2002;26:161-69.

14. Jensen WA, Fall MZ, Rooney J, Kordick DL, Breitschwerdt EB. Rapid identification and differentiationof Bartonella species using a single-step PCR assay. J Clin Microbiol. 2000;38:1717-22.

15. Guccion JG, Gilbert CL, Ortega LG, Hadfield TL. Cat scratch disease and acquired immunodeficiencydisease: diagnosis by transmission electron microscopy. Ultrastruct Pathol. 1996;20:195-202.

MAILING ADDRESS:Paulo Eduardo Neves Ferreira VelhoDepartamento de Clínica Médica da Faculdadede Ciências Médicas da Universidade Estadualde Campinas – FCM / Unicamp Cidade Universitária Zeferino Vaz, s/n.13083-970 - Campinas - SP - BrazilTel./Fax: +55 (19)3289-4107E-mail: [email protected]

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